RESEARCH ARTICLE 5135
Development 138, 5135-5146 (2011) doi:10.1242/dev.067801
© 2011. Published by The Company of Biologists Ltd
Combinatorial roles for BMPs and Endothelin 1 in patterning
the dorsal-ventral axis of the craniofacial skeleton
Courtney Alexander1, Elizabeth Zuniga2, Ira L. Blitz1, Naoyuki Wada1,*, Pierre Le Pabic1, Yashar Javidan1,
Tailin Zhang1, Ken W. Cho1, J. Gage Crump2 and Thomas F. Schilling1,‡
SUMMARY
Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions
with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal
arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive
transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in
surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a
[Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC
migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the
ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal
signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch
skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding
reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of
either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are
established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1.
This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning,
but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.
INTRODUCTION
How do cartilages and bones acquire their distinct threedimensional shapes and sizes during development? The
craniofacial skeleton derives from cranial neural crest (NC) cells
that migrate from the neural tube into the pharyngeal arches.
Although cranial NC cells acquire their anterior-posterior identities
(mandibular versus hyoid arch) prior to migration, patterning
events at post-migratory stages largely determine the
morphogenesis of individual skeletal elements (Trainor and
Krumlauf, 2000; Schilling et al., 2001; Knight and Schilling, 2006).
Overlapping expression domains of transcription factors of the Dlx
family, as well as Hand2 (Clouthier et al., 2010) pattern each
pharyngeal segment along its dorsal-ventral (D-V) axis (equivalent
to the proximal-distal axis in mice). These early domains are
induced by signals from surrounding epithelia, including the
endoderm and ectoderm that line each arch (David et al., 2002;
Trainor and Krumlauf, 2000); in the mandibular arch, for example,
they roughly determine ventral (e.g. lower jaw or mandible),
intermediate (jaw joint) and dorsal (e.g. upper jaw) identities.
However, how multiple signals are integrated to determine
craniofacial patterning remains largely unknown.
1
Department of Developmental and Cell Biology, University of California, Irvine,
CA 92697, USA. 2Eli and Edythe Broad Institute for Regenerative Medicine and Stem
Cell Research, Department of Cell and Neurobiology, University of Southern
California, Los Angeles, CA 90033, USA.
*Present address: Applied Biological Science, Faculty of Science and Technology,
Tokyo, University of Science, Noda, Chiba, 278-8510, Japan
‡
Author for correspondence ([email protected])
Accepted 15 September 2011
Endothelin 1 (Edn1), a small, secreted ligand produced by
ventral pharyngeal ectoderm, mesoderm and endoderm, induces
ventral and intermediate skeletal identities in the arches (Thomas
et al., 1998; Clouthier et al., 2000; Miller et al., 2000; Miller et al.,
2003). In mice and zebrafish deficient in Edn1 or its receptor
Ednra, the mandible is reduced and fuses with the upper jaw
instead of forming a joint (Ozeki et al., 2004; Miller et al., 2000;
Maemura et al., 1996; Clouthier et al., 1998; Yanagisawa et al.,
1998b). Disruptions of Edn1 target genes also cause ventral- and
intermediate-specific defects: (1) mice with a combined loss of
Dlx5/6 have reduced/transformed mandibles (Beverdam et al.,
2002; Depew et al., 2002); (2) hand2 mutant zebrafish and mice
have reduced mandibles (Miller et al., 2003; Yanagisawa et al.,
2003); and (3) zebrafish deficient in dlx3b/4b/5a (Talbot et al.,
2010), as well as nkx3.2 (Miller et al., 2003), have fused jaw joints.
Edn1 signaling also restricts the Notch ligand, Jag1b, dorsally,
suggesting that the two signals act in opposition to subdivide DV
domains within the arches (Zuniga et al., 2010). Edn1 may act as
a morphogen, activating ventral and intermediate genes at different
concentrations (Kimmel et al., 2003; Kimmel et al., 2007).
However, uniform application of Edn1 protein, as well as loss of
jag1b function, can restore many aspects of DV patterning in
zebrafish edn1–/– mutants (Miller et al., 2000; Zuniga et al., 2010).
This suggests that the spatial localization of Edn1 is not crucial and
that other ventralizing signals must interact with Edn1 to control
DV patterning (Clouthier et al., 2010).
Bone morphogenetic proteins (BMPs) are likely to be one such
instructive signal in DV patterning. In both zebrafish and mice,
ventral arch ectoderm expresses Bmp4 (Aberg et al., 1997; Vainio
et al., 1993; Martinez-Barbera et al., 1997), cranial NC expresses
Bmp2 (Nie et al., 2006), and the pharyngeal endoderm/ectoderm
DEVELOPMENT
KEY WORDS: Neural crest, Pharyngeal arch, Branchial arch, Craniofacial, Xenopus, Zebrafish
5136 RESEARCH ARTICLE
MATERIALS AND METHODS
Generation of a Bre:GFP transgenic line
Our 21 bp BMP responsive element (Bre) contains core Smad1- and
Smad4-binding elements derived from the promoter region of the Xenopus
Xvent2 gene, separated by a 5 bp nucleotide spacer (von Bubnoff et al.,
2005; Yao et al., 2006). By testing single and multimerized versions of the
Bre, we found a luciferase construct containing five tandem elements (5XBre) that is much more sensitive than a single element (data not shown).
This 5x-Bre was placed upstream of a minimal Xenopus Id3 promoter
driving GFP (supplementary material Fig. S1). Three stable Tg(Bre:GFP)
transgenic lines (p60, p69 and p77) were independently isolated and all
showed similar expression patterns (supplementary material Fig. S1). We
chose Tg(Bre:GFP)p77 for further analysis because it showed the brightest
GFP expression.
mRNA and MO injections
xBmp4 and xNoggin (xNog) mRNAs were transcribed using
mMessageMachine SP6 (Applied Biosystems) after linearization with XhoI
and EcoRI, respectively. mRNA was diluted to 50-500 pg/nl in diethyl
pyrocarbonate (DEPC)-treated water and 0.5 nl was pressure injected into
single-cell zebrafish embryos. Embryos were fixed with 4%
paraformaldehyde (PFA) at 6 hours post-fertilization (hpf) for
immunostaining with anti-GFP (Abcam) and anti-pSmad1/5/8 (Millipore).
Heat shock conditions
Zebrafish embryos obtained from Tg(hsp70I:dnBmpr1a-GFP)w30 (Pyati et
al., 2005) heterozygous adult incrosses were heated for 1 hour at 39°C in
a PCR machine and subsequently raised in a 28.5°C incubator. After heat
shock, GFP+ fluorescing embryos were sorted from GFP–, nonfluorescent
siblings and either fixed for in situ hybridization and immunostaining 1-2
hours later or raised to 4-5 days post fertilization (dpf) for Alcian staining
of cartilage (Walker and Kimmel, 2007). For Tg(hsp70I:Gal4; UAS:Bmp4)
and Tg(hsp70I:Gal4; UAS:Edn1) activations, embryos were placed in a
programmable incubator at 40°C for 4-8 hours, as indicated, and then
returned to 28.5°C. For shorter Tg(hsp70I:Gal4; UAS:Bmp4) treatments,
embryos were placed in 40°C pre-warmed embryo medium (EM) at 21 hpf
and transferred to 28.5°C EM after 3 minutes.
Bead implantation
Affi-gel blue beads (Bio-Rad) were incubated in a solution of human
recombinant BMP4 homodimer (R&D Systems), BMP4/7 heterodimer (R&D
Systems), NOGGIN (R&D Systems) or EDN1 (Sigma), or bovine serum
albumin (BSA) diluted in phosphate-buffered saline (PBS) overnight at 4°C
and just prior to implantation placed at 37°C for 1 hour. Zebrafish embryos at
22-24 hpf were mounted in 3-4% methylcellulose and a small slit was made
with a tungsten needle directly behind the eye above the presumptive first
pharyngeal arch. A protein-coated bead was inserted into the slit using the flat
edge of the tungsten needle. Embryos were transferred to embryo medium for
recovery and fixed in 4% PFA either at stages of DV patterning (28-36 hpf)
for in situ hybridization or at larval stages (4-5 dpf) for cartilage staining.
Protein injection
Zebrafish embryos at 22-24 hpf were mounted in 1% low melt agarose and
a 0.5 nl droplet of a 1.0 mg/ml solution of human EDN1 protein (SigmaAldrich), diluted in water, was pressure injected through a glass
microelectrode into the arch primordium. Embryos were either fixed in 4%
PFA for in situ hybridization (20-36 hpf) or allowed to develop to 4-5 dpf
for cartilage staining.
Phenotypic analysis
Zebrafish embryos (4-5 dpf) were stained overnight using an acid-free Alcian
Blue solution (Walker and Kimmel, 2007). Embryos were then rehydrated
into PBS, bleached in 1% KOH/3% H2O2, cleared in 0.05% trypsin and
stored in 70% glycerol. Cartilage was dissected and flat mounted as
previously described (Javidan and Schilling, 2004). Colorimetric (Thisse et
al., 1993) or two-color fluorescent (Zuniga et al., 2010) in situ hybridizations
were performed. An antisense probe for GFP mRNA to detect Tg(Bre:GFP)
expression was synthesized using T3 polymerase after linearization with
HindIII. Other antisense probes included dlx2a (Akimenko et al., 1994),
dlx6a, dlx5a and dlx3b (Akimenko et al., 1994), hand2 (Yelon et al., 2000),
msxe (Miller et al., 2003), nkx3.2 (Miller et al., 2003), and jag1b (Zuniga et
al., 2010). Immunolabeling was performed with anti-GFP and antipSmad1/5/8 antibodies, and detected using 1:500 DyLight 488 donkey antichick (Jackson ImmunoResearch) and 1:200 Alexa Fluor 564 goat anti-rabbit
(Invitrogen). Embryos were fixed with 4% PFA and processed as previously
described (Westerfield, 2000).
RESULTS
A BMP-responsive reporter reveals direct
responses in the ventral pharyngeal arches
Loss-of-function mutations in Bmp7 and Bmpr1 in mice, as well as
conditional deletions of Bmp4 in arch epithelia or Smad4 within the
cranial neural crest (NC), cause craniofacial defects, including
DEVELOPMENT
expresses bmp5 and bmp7a/b (Holzschuh et al., 2005). Like Edn1,
BMPs induce transcription factors such as Msx1 ventrally (Tucker
et al., 1998a) and restrict expression of Nkx3.2 dorsally to specify
the jaw joint (Wilson and Tucker, 2004). Bmp4 also restricts Fgf8
and Barx1 expression dorsally in the oral epithelium and
mesenchyme, respectively, to determine tooth type, which restricts
incisors ventrally and molars dorsally (Tucker et al., 1998b; Tucker
et al., 1999). Conditional removal of Bmp4 in the arch ectoderm
and ventral endoderm (Liu et al., 2005), as well as removal of Alk2
(a type 1 BMP receptor) in NC cells (Dudas et al., 2004), disrupts
formation of the mandible. Conditional removal of Smad4, a key
regulator of downstream BMP signaling, in cranial NC cells leads
to the arrest of both incisors and molars in the mandibular arch (Ko
et al., 2007).
Despite this body of evidence indicating requirements in
craniofacial development, the precise roles of BMPs in DV
patterning of the arches remain unclear. Other members of the Tgf
superfamily (e.g. Gdf and Nodal proteins) also influence craniofacial
and skeletal development (Oka et al., 2007; Chai et al., 2003). BMP
receptors phosphorylate Smads 1/5/8 (pSmad1/5/8), which bind in a
complex with Smad4 to a Smad-binding element in the promoter
regions of BMP target genes. The typical method of detecting BMP
signaling in developing animals, including zebrafish, relies on the
detection of pSmad1/5/8 by immunostaining. Although useful, this
approach is: (1) not a direct readout of transcriptional activation by
BMPs; and (2) unsuitable for in vivo imaging as detection of
phosphorylated Smad activity requires tissue fixation.
Here, we describe a new transgenic reporter line of zebrafish
containing a BMP-response element driving GFP, Tg(Bre:GFP),
that specifically responds to endogenous BMP signaling activity
and allows in vivo imaging of BMP signaling in developing
zebrafish embryos. Using Tg(Bre:GFP) embryos, we show direct
BMP responses in the ventral NC and surrounding epithelia of the
pharyngeal arches. Using a dominant-negative BMP receptor
transgenic, Tg(hsp70I:dnBmpr1a-GFP) (Pyati et al., 2005), we
show that blocking BMP signaling soon after NC migration causes
severe ventral (and intermediate) defects in the mandibular and
hyoid arches, as well as disrupting the trabecular cartilages of the
palate. Ventral expansion of jag1b expression in these BMP
signaling-deficient embryos suggests a ventral-to-dorsal
transformation. Conversely, overexpressing BMPs leads to specific
transformations of dorsal and intermediate structures that are
suggestive of a dorsal-to-ventral transformation. Initially, BMP
induces Edn1 and the two signals appear redundant in DV
patterning. However, effects of Edn1 later become restricted to the
intermediate, joint-forming domain, whereas BMPs induce more
ventral (mandible) and anterior (palate) domains, revealing a novel
temporal component to patterning of the craniofacial skeleton.
Development 138 (23)
BMP and Edn1 in craniofacial development
RESEARCH ARTICLE 5137
Fig. 1. Expression of Tg(Bre:GFP) in
the pharyngeal arches. (A-C)GFP
fluorescence in living Tg(Bre:GFP)
transgenic embryos, lateral views,
anterior towards the left. (D-F)Lateral
views of the arches at higher
magnification. Confocal slices of
Tg(Bre:GFP) (D) and Tg(sox10:lyntdTomato) (E) double transgenics. The
two channels are merged in F,
demonstrating direct BMP responses in
NC and in the stomodeum (arrow).
(G-I)Adjacent transverse sections through
the hindbrain and arches stained with
anti-GFP (G,I) and anti-pSmad1/5/8 (H),
revealing BMP responses in the NC and in
surrounding endoderm (en) and
ectoderm (ec). (A)16 hpf. (B)24 hpf.
(C)48 hpf. (D-F)28 hpf. (G-I)30 hpf. cn,
commissural neurons; D, dorsal; de,
dorsal eye; ec, ectoderm; en, endoderm;
I, intermediate; le, lens; nc, neural crest;
pa, pharyngeal arches; rp, roofplate; s,
somites; V, ventral. Scale bars: 100mm.
Optical sections through the arches also revealed strong
Tg(Bre:GFP) expression in arch epithelia, including the
stomodeum and endoderm (Fig. 1F). To confirm this, we compared
Tg(Bre:GFP) expression (detected with an anti-GFP antibody) with
pSmad1/5/8 immunostaining. In transverse sections at 30 hpf, both
Tg(Bre:GFP) and pSmad1/5/8 were detected in arch epithelia
(endoderm and ectoderm), as well as in NC cells throughout the
arches (Fig. 1G-I). These results suggest that BMPs signal directly
to NC cells throughout the mandibular and hyoid arches, as well as
to surrounding arch epithelia, and that the strongest response is
localized ventrally during the establishment of DV skeletal
patterning.
BMP signaling is required after NC migration for
lower jaw and palate development
BMP signaling is implicated in NC induction, migration,
proliferation and survival, as well as patterning and differentiation
of the facial skeleton and teeth (Christiansen et al., 2000; Knecht
and Bronner-Fraser, 2002; Dudas et al., 2004; Liu et al., 2005).
Previous conditional mutants in mice to remove BMP signaling in
NC cells could not distinguish between requirements in
premigratory versus postmigratory NC (Ko et al., 2007). To
address this issue, we used a transgenic line of zebrafish that
expresses a heat shock-inducible, dominant-negative type 1 BMP
receptor 1a [Tg(hsp70I:dnBmpr1a-GFP)], hereafter referred to as
dnBmpr (Pyati et al., 2005). Homozygous dnBmpr transgenics
(identified by strong GFP expression), heated for 1 hour at 39°C,
lost pSmad1/5/8 immunostaining in the arches by 2 hours post-heat
shock and this loss was still apparent up to 6 hours post-heat shock
(supplementary material Fig. S3) when compared with controls
(GFP-negative siblings) heat shocked in a similar manner. Analyses
of pharyngeal cartilage formation at larval stages were carried out
DEVELOPMENT
reduced mandibles and/or cleft lips/palates (Dudas et al., 2004; Liu
et al., 2005; Ko et al., 2007). Because the mandible and portions of
the palate develop from the mandibular arch (pharyngeal arch 1)
during embryogenesis, we hypothesized that NC cells in this arch
respond directly to BMPs. Previous analysis using a BMP response
element (Bre) driving GFP (Bre:GFP) expression in Xenopus laevis
embryos detected BMP responses in the pharyngeal region (von
Bubnoff et al., 2005). We took advantage of this construct to
develop a new zebrafish reporter line, Tg(Bre:GFP), that marks
BMP signaling activity (Fig. 1). In zebrafish embryos,
Tg(Bre:GFP) was first detected on the ventral side of the gastrula
at 6 hpf, and during segmentation (14-24 hpf) marked the dorsal
eye, roof plate of the presumptive mid/hindbrain and somites
(supplementary material Fig. S1), all regions with known
functional roles for BMP signaling. Tg(Bre:GFP) expression was
also: (1) correlated with immunostaining for pSmad1/5/8; (2)
induced by the addition of exogenous Bmp4 mRNA or human
recombinant BMP4/7 protein; and (3) reduced by BMP signaling
inhibitors, such as Noggin (Nog), indicating that our reporter is
both specific and sensitive (supplementary material Fig. S2). By 16
hpf, GFP expression was detected in the pharyngeal arches (Fig.
1A) and this persisted throughout embryogenesis and into larval
stages (Fig. 1B,C; supplementary material Fig. S1), when
expression increased in pharyngeal muscles. Thus, our reporter
confirmed direct BMP signaling in the pharyngeal arches
throughout embryonic arch morphogenesis and differentiation.
To determine which NC cells within the arches respond directly,
we created double transgenics for the Tg(Bre:GFP) and
Tg(sox10:lyn-tdTomato), which marks migrating cranial NC cells
(Fig. 1D-F). Confocal microscopy revealed co-expression of the
two transgenes throughout arch NC at 28 hpf, with the strongest
expression in ventral domains of arches 1 and 2 (Fig. 1D,F).
5138 RESEARCH ARTICLE
Development 138 (23)
after a careful time course of heat shocks between 12 and 36 hpf,
thus revealing a dnBmpr-sensitive period between 15 and 22 hpf.
Taking into account at least a 2-hour delay before complete loss of
BMP signaling, this suggests a crucial period for BMP signaling in
pharyngeal patterning from 17-24 hpf (Fig. 2A).
Homozygous dnBmpr embryos heat shocked between 16-18 hpf
showed severe reductions of ventral cartilages and intermediate
elements (e.g. joints) in both the mandibular (first) and hyoid
(second) arches when compared with heat-shocked controls (Fig.
2B-D). Ventral mandibular (Meckel’s cartilage, Mc) and hyoid
(ceratohyal, Ch) elements were reduced in size or occasionally lost
completely with high expressivity: 92% (mandibular) and 89%
(hyoid) (n28). The first arch joint was frequently also fused (92%,
n28) and the retroarticular process (ra, arch 1), symplectic (Sy,
arch 2) and interhyal (Ih, arch 2), all intermediate domain
derivatives, were consistently absent (Fig. 2D,D⬘). Dorsal elements
were also affected in heat-shocked dnBmpr embryos. The
hyomandibular (Hm, dorsal arch 2, arrows in Fig. 2C-D⬘) retained
its respective shape with the occasional reduction in size, as
previously reported for edn1–/– and hand2–/– mutants (Miller et al.,
2000; Miller et al., 2003), but the palatoquadrate (Pq, dorsal arch
1) was more severely affected, either reduced or more rod-like in
shape. Consistent with a dose-dependent requirement for Bmpr1a,
heterozygous dnBmpr transgenics (identifiable by their weak GFP
expression) heat shocked at the same stages showed mild
reductions in Mc and lacked the mandibular joint, but dorsal
elements were well formed (Fig. 2C,C⬘). Heat-shocked embryos
showed no gross reductions in arch NC, as assayed in
Tg(hsp70I:dnBmpr1a-GFP);Tg(sox10:lyn-tdTomato)
double
transgenics and the number of cells undergoing apoptosis was
similar in heat shocked and control embryos (supplementary
material Fig. S4). Thus, BMP signaling is required between 16 and
24 hpf for patterning and/or differentiation of craniofacial
cartilages, particularly the ventral and intermediate domain-derived
cartilages of the first two arches.
Strikingly, 92% of dnBmpr embryos heat shocked at 16-18 hpf
partially or completely lost the trabecular cartilages of the palate but
retained a midline ethmoid plate, and defects were stronger in
homozygotes than heterozygotes (Fig. 2E-G). This was intriguing
because we had previously shown that these cartilages arise from
separate populations of NC cells that have different migratory paths
from their relatives in the pharyngeal arches (Wada et al., 2005).
Ethmoid precursors migrate anteriorly between the eyes, whereas
trabecular precursors migrate just posterior to eye and at 20-24 hpf
reside just dorsal and anterior to the oral epithelium of the
stomodeum. Both the stomodeum and the NC cells in this location
express Tg(Bre:GFP) (see Fig. 1D,F). To determine whether
reducing BMP signaling alters stomodeal development, we examined
pitx2ca and fgf8a expression in heat-shocked dnBmpr embryos and
found occasional reductions in pitx2ca and variable expansion of the
DEVELOPMENT
Fig. 2. Requirements for BMP signaling
in pharyngeal cartilage and palate
development. (A)Histogram quantifying
the frequency of defects in different skeletal
elements caused by heat shocking
Tg(hs:dnBmpra1-GFP) embryos at different
stages between 15 and 36 hpf. (B-G)Alcian
Blue stained cartilages of 5 dpf larvae,
dissected and flat-mounted, anterior towards
the left. (B-D)Pharyngeal cartilages of
control (B), moderate (C) and severely
affected (D) Tg(hs:dnBmpra1-GFP)
transgenics, heat shocked at 16-18 hpf,
lateral views. (B⬘-D⬘) Diagrams of cartilage
elements in arch 1 (mandibular, Mc and Pq)
and arch 2 (hyoid, Ch, Ih and Hm)
corresponding to cartilages in B-D. Lateral
views, anterior towards the left. Colors
indicate dorsal (green), intermediate (yellow)
and ventral (pink) elements. Arrows indicate
dorsal arch 2 elements; asterisks indicate
fused joints in arch 1; arrowheads indicate
joints in arch 2. (E-G)Neurocranial cartilages
of the palate, ventral view: control (E),
moderate (F) and severe (G). abc, anterior
basicranial commissure; Ch, ceratohyal; e,
ethmoid plate; Hm, hyomandibular; Ih,
interhyal; Mc, Meckels cartilage; pc,
parachordals; Pq, palatoquadrate; ptp,
pterygoid process; ra, retroarticular process
of Meckel’s cartilage; Sy, symplectic; tr,
trabeculae. Scale bars: 100mm.
BMP and Edn1 in craniofacial development
RESEARCH ARTICLE 5139
Fig. 3. BMP signaling is required for ventral cell specification in the arches. (A-P)Whole-mount in situ hybridization to detect expression of
genes involved in DV arch patterning, lateral views, anterior towards the left in controls (A,C,E,G,I,K,M,O) and Tg(hsp70I:dnBmpra1-GFP)
transgenics heat shocked at 16-18 hpf (B,D,F,H,J,L,N,P). dlx3b (A,B), dlx6a (C,D) and hand2 (E,F) expression is reduced at 28 hpf. nkx3.2 (G,H)
expression is reduced and jag1b expression (I,J) expands ventrally at 36 hpf (arrows). msxe expression is lost at 30 hpf (K,L). Arches 1 and 2 are
numbered. (M-P)Two-color fluorescent in situs to detect dlx3b (M,N, red) or msxe (O,P, red) simultaneously with hand2 (green). Scale bar: 100mm.
BMP signaling is required to promote ventral and
repress dorsal arch identities
Skeletal defects in BMP signaling-deficient embryos are
reminiscent of mutants in both fish and mice that disrupt DV
patterning of the arches (e.g. Edn1–/–, EdnrA–/– and Dlx5–/–;
Dlx6–/–) (Miller et al., 2000; Depew et al., 2002; Beverdam et al.,
2003; Ozeki et al., 2004; Nair et al., 2007) and several models have
been proposed in which BMPs promote ventral (distal) arch
identities. To investigate this model in more detail, we examined
changes in expression patterns of genes required for establishing
different DV domains within the arches. dnBmpr transgenic
embryos were analyzed at 30 hpf after heat shocking at 16 hpf (Fig.
3). dlx2a is expressed throughout the DV axis of the arches and its
expression was not disrupted in heat-shocked dnBmpr embryos
(supplementary material Fig. S6). By contrast, dlx3b and dlx6a
expression are intermediate along the DV axis, with dlx3b
expression nested within the dlx6a expression domain (Talbot et
al., 2010), and both were reduced in heat shocked dnBmpr
transgenics (dlx3b: 66%, n12/18; dlx6a: 69%, n27/39) (Fig. 3AD,M,N; supplementary material Fig. S6A,B). Within this
intermediate domain, nkx3.2 expression in joint progenitors within
the mandibular arch was also severely reduced in these embryos
(Fig. 3G,H; supplementary material Fig. S6C,D), as was expression
of msxe in a more ventral-intermediate domain (80%, n8/10) (Fig.
3K,L). Expression of hand2 in the ventral-most arch was also
severely reduced (88%, n32/36), with the remaining expression
largely confined to epithelia at arch borders (Fig. 3E,F,M-P),
similar to edn1–/– mutants. Based on these findings, we conclude
that BMP signaling is required for specifying ventral and
intermediate domains within the arches by regulating the
expression domains of these transcription factors.
jag1b is a Notch ligand required for dorsal arch identities, and
its mRNA is normally restricted to a dorsal-posterior domain within
each arch. Importantly, jag1b expression was expanded ventrally
throughout much of the intermediate and ventral domains in heatshocked dnBmpr transgenics (100%, n5/5) (Fig. 3I,J). These
results suggest that loss of BMP signaling during arch development
leads to a loss of ventralizing signals, and expansion of dorsalizing
signals, resulting in partial transformations of ventral/intermediate
domains to a more dorsal identity (see below).
BMPs are sufficient to induce ventral arch
identities
In a complementary gain-of-function approach, we tested the
effects on skeletal development and DV patterning of increasing
BMP signaling locally within the first arch at 22-24 hpf. Beads
soaked in human recombinant BMP4 protein or BMP4/7
heterodimers (chosen because of their conserved expression in the
arches across species) were implanted just behind the eye and the
embryos raised for cartilage analysis 3 days later. Although BSAsoaked control beads had no effect on cartilage differentiation or
arch morphology (Fig. 4A,C), beads soaked in human
DEVELOPMENT
fgf8a expression domain (supplementary material Fig. S5) (Hu et al.,
2003; Eberhart et al., 2006). These results suggest that, in addition
to its roles in the lower jaw and jaw joint, BMP signaling also
induces trabecular/palate formation and controls expression of early
developmental regulatory genes in the oral ectoderm.
5140 RESEARCH ARTICLE
Development 138 (23)
Fig. 4. Exogenous BMP is sufficient to induce ventral arch identity. (A-D,F-H) Alcian Blue-stained cartilages of 4-5 dpf larvae, dissected and
flat-mounted, anterior towards the left. (I-N)Whole-mount in situ hybridization at 30 hpf, lateral views, anterior towards the left. (A,B)Pharyngeal
cartilages of a control (A) and an embryo implanted behind the eye at 20 hpf with a bead soaked in 20mg/ml of human recombinant BMP4/7
heterodimers (B). (C,D)Isolated cartilages of the mandibular (1) and hyoid (2) arches: control (C); BMP4/7 bead-implanted (D). Grey arrowheads in
B,D indicate duplicated Mc cartilages. (E)Histogram of phenotype frequency in bead-implanted embryos depending on BMP4/7 concentration.
(F-H)Neurocranial cartilages: control (F); BMP4/7 bead-implanted (G); heat-shocked Tg(hsp70I:Gal4;UAS:Bmp4) (H). Black arrowheads indicate
ectopic cartilages. (I,J)dlx6a expression slightly expands dorsally (arrow) in response to a BMP4/7-soaked bead (J). (K,L)dlx3b expression does not
change when BMP4/7-soaked beads are implanted. (M,N)hand2 expression also expands dorsally (arrow) in response to BMP4/7 protein. Asterisks
and blue circles indicate beads. 1, mandibular arch; 2, hyoid arch; Ch, ceratohyal; e, ethmoid plate; Hm, hyomandibular; Mc, Meckel’s cartilage;
Mc’, ectopic Meckel’s cartilage; Pq, palatoquadrate; tr, trabeculae. Scale bars: 100mm.
with BSA controls (Fig. 4I,J; 100%, n6) whereas expression of
dlx3b appeared unaffected (Fig. 4K,L; 100%, n6). By contrast,
hand2 expression, which is normally restricted to the ventral
domain, was strongly induced throughout the first arch in response
to exogenous BMP4/7 (Fig. 4M,N; 60%, n5). Similar results were
obtained in heat-shocked Tg(hsp70I:Gal4;UAS:Bmp4) embryos
(Zuniga et al., 2011). Taken together with the loss-of-function data,
these results suggest that hand2 is particularly sensitive to BMP
signaling in the ventral arches, whereas expression of dlx6a and
other intermediate-domain genes appears less sensitive.
Stage-specific interactions between BMP and Edn1
signaling in arch patterning
Edn1 signaling is required for ventral arch development and
expression of hand2, dlx3b, dlx6a and nkx3.2, but recent models
suggest that additional signals must work together with Edn1 to
establish DV pattern (Clouthier et al., 2010). To determine whether
BMP and Edn1 signals interact genetically in arch development,
we injected 2.5 ng of an edn1 morpholino (edn1-MO) into dnBmpr
transgenics, heat shocked them and assayed cartilage morphology
(supplementary material Fig. S7). Either edn1-MO or heterozygous
dnBmpr+/– transgenics alone caused reductions in ventral cartilages
and mandibular joint fusions. By contrast, ventral elements were
completely absent in 66% (n9) of double mutants/morphants.
These results suggest that BMP and Edn1 signaling synergize to
promote ventral arch development.
We next examined epistatic relationships between Edn1 and
BMP signaling. edn1 expression was severely reduced or lost in
dnBmpr embryos heat shocked from 16-18 hpf (Fig. 5B), and
DEVELOPMENT
recombinant BMP4 homodimers (10-25 mg/ml) or BMP4/7
heterodimers (which were more potent: 1-10 mg/ml) induced rodshaped cartilage elements that resembled Mc (Fig. 4B,D). This
was more common at lower concentrations and was typically
located dorsal to Mc, in the position of the normal pterygoid
process (Ptp), and occasionally fused to the endogenous Mc (not
shown). The dorsal Pq was also consistently reduced or absent
and its ventral aspect occasionally acquired an Mc-like
morphology in BMP-implanted embryos (Fig. 4B,D). Trabecular
cartilages of the palate were also expanded or malformed in
response to exogenous BMPs (Fig. 4G). Similar arch and
trabecular malformations were obtained with global
overexpression of Bmp4 using the transgenic line
Tg(hsp70I:Gal4;UAS:Bmp4) – generated by crossing a line
containing hsp70I driving GAL4 expression (hsp70I:Gal4) and
one containing Bmp4 driven by the UAS promoter (UAS:Bmp4)
heat shocked at 20-24 hpf. Based on the position and morphology
of the rod-shaped cartilages we interpreted this as evidence of a
partial DV transformation of the pterygoid process (and possibly
the ventral aspect of Pq) to a more-ventral, Mc-like morphology
in the first arch. These results support the hypothesis that BMPs
are sufficient to promote ventral arch development at the expense
of dorsal arch development and to pattern the palate.
To determine whether these effects of exogenous BMPs on the
skeleton reflect earlier changes in DV patterning we examined gene
expression at 30 hpf in embryos implanted with human
recombinant BMP4/7-soaked beads. Expression of dlx6a in its
intermediate domain was slightly expanded dorsally in the first
arch in response to beads soaked in 10 mg BMP4/7 when compared
BMP and Edn1 in craniofacial development
RESEARCH ARTICLE 5141
Fig. 5. BMPs regulate Edn1 expression.
Projections of double fluorescent in situ
hybridization, lateral views, anterior towards the
left. (A-C)dlx2a (red) and edn1 (green) in wild-type
(A), heat shocked Tg(hsp70I:dnBmpr1a-GFP) (B) and
heat shocked Tg(hsp70I:Gal4;UAS:Bmp4) (C)
embryos. (D-F)dlx2a (red) and bmp4 (green) in
wild-type (D), edn1–/– mutant (E) and heat shocked
Tg(hsp70I:Gal4;UAS:Edn1) (F) embryos. Scale bar:
50mm.
As Hand2 expression requires Edn1, we hypothesized that
induction occurs at an earlier stage and becomes BMP-dependent
after 24 hpf. In zebrafish, efficient ventralization with EDN1
protein injections only occurs within a narrow time window around
24 hpf (Miller et al., 2000; Kimmel et al., 2007). Thus, we injected
0.5 mg EDN1 protein at several timepoints between 18 and 36 hpf
in heat-shocked dnBmpr transgenics (heat shocked at 16 hpf).
However, in all cases these failed to cause any detectable increase
in the pattern or levels of hand2 expression in the arches (data not
shown), and this correlated with a failure to completely rescue Mc
(see Fig. 6). Similarly, Tg(hsp70I:Gal4;UAS:Edn1) transgenics
heat shocked between 16 and 28 hpf failed to rescue hand2
expression in BMP-deficient embryos (Zuniga et al., 2011). These
results confirm the hand2 expression is BMP-dependent at both
early and later stages of arch development.
DISCUSSION
In this study, we show that BMPs act upstream and in parallel to
Edn1 to promote ventral skeletal fates in the pharyngeal arches,
with BMPs having an Edn1-independent role in palate
development (Fig. 8). Initially, BMPs induce Edn1 expression
ventrally and both signals repress Jag1b dorsally to induce ventral
(lower jaw) and intermediate (joint) skeletal fates in the mandibular
arch (Fig. 8A). Later, the roles of BMPs and Edn1 become spatially
distinct, with BMPs inducing the ventral-most fates within the arch
while Edn1 induces more intermediate fates (Fig. 8B; Zuniga et al.,
2011). Our model helps reconcile the fact that BMPs and Edn1 are
both required for ventral arch development. It is consistent with
data showing that BMP and Edn1 signaling regulate some common
targets, such as Msx1, and not others. It also helps explain why
defects in either BMP or Edn1 alone fail to completely eliminate
or transform the mandible, as they act together in a DV patterning
network.
Spatial and temporal specificity in BMP signaling
during craniofacial development
Previous studies have shown that removing Bmp4 in cranial epithelia
(or Smad4 in NC cells) in mice causes lower jaw defects (Liu et al.,
2005; Ko et al., 2007). However, BMPs have been implicated in NC
induction, proliferation, survival and differentiation (Christiansen et
al., 2000; Aybar and Mayor, 2002; Knecht and Bronner-Fraser,
2002), and problems with any of these might indirectly cause such
DV defects. We provide evidence for a more direct role in patterning
by: (1) defining a time window of BMP activity after NC migration;
and (2) demonstrating that BMPs specify ventral and intermediate
domains at the expense of dorsal. We first detect Tg(Bre:GFP)
DEVELOPMENT
expanded dorsally in Tg(hsp70I:Gal4;UAS:Bmp4) embryos heat
shocked at 20-24 hpf (Fig. 5C). By contrast, we detected no change
in bmp4 expression in edn1–/– mutants compared with wild type
(Fig. 5D,E) (Miller et al., 2000), or in embryos overexpressing
Edn1 using Tg(hsp70I:Gal4;UAS:Edn1) double transgenics heat
shocked at 20-24 hpf. These results suggest that BMPs act, at least
in part, upstream of edn1 in DV arch patterning.
To address this further, we attempted to rescue deficiencies in
BMP signaling by overexpressing Edn1, and vice versa.
Microinjection of human recombinant EDN1 protein into the
extracellular space of the arch mesenchyme rescued arch
development in heat-shocked dnBmpr transgenics in a dosedependent manner (Miller et al., 2000; Kimmel et al., 2007).
Injection of 0.5 mg EDN1 protein over a time series from 24-31 hpf
(8-15 hours post-heat shock at 16 hpf) partially restored Mc and Ch
cartilages and the mandibular joint (Fig. 6A-D), but did not restore
development of more dorsal arch cartilages or the trabecular
cartilages of the palate (Fig. 6F-H). Injection of EDN1 after 31 hpf
failed to rescue the phenotype (Fig. 6E), suggesting there is a
window from 24-31 hpf during which EDN1 is sufficient to pattern
ventral and intermediate cartilages. Rescue of Mc was less frequent
whereas rescue of Ch in arch 2 was typically the most robust.
Similarly, overexpression of Bmp4 by heat shocking
Tg(hsp70I:Gal4;UAS:Bmp4) double transgenics at 20 hpf rescued
ventral arch development in edn1–/– mutants (Fig. 6I-K). However,
rescue was restricted to ventral cartilages and joints remained
fused. In addition, rescue occurred only with short heat shocks (13 minutes), and higher amounts of BMP, either with longer heat
shocks or implantation of human recombinant BMP4/7-soaked
beads at 22-24 hpf, failed to rescue the skeleton (data not shown;
3/3) or the expression of dlx5b (supplementary material Fig. S8).
These results suggest that BMPs can promote ventral skeletal fates
in the arches in an Edn1-independent manner.
To investigate this further, we examined the ability of Edn1 to
rescue gene expression in ventral and intermediate domains of
the arches in BMP-deficient embryos. Injections of EDN1
protein restored dlx5a (44%, n9) and dlx6 (83%, n6), and
msxe (66%, n15) to their normal patterns of expression (Fig.
7G-O) and strongly induced dlx3b (50%, n8) throughout much
of the DV extent of the first and second arches (Fig. 7D-F).
Surprisingly, however, restoring Edn1 expression failed to rescue
hand2 expression (0%, n16) or to completely rescue Mc
cartilage to its normal size. This suggests that Edn1 can promote
intermediate domain Dlx gene expression and ventral arch
skeletal fates in a BMP-independent manner but requires BMP
to induce hand2.
5142 RESEARCH ARTICLE
Development 138 (23)
expression (and Smad1/5/8 phosphorylation) in the mandibular arch
in zebrafish at 16 hpf and prominently in arch NC cells by 24 hpf.
By 28 hpf, Tg(Bre:GFP) expression reveals at least two distinct
states within the arches, high BMP signaling in ventral domains and
low signal in intermediate and dorsal domains, as opposed to a BMP
signaling gradient. Consistent with this, heat-shocking dnBmpr
transgenics between 16 and 18 hpf causes strong reductions in
ventral cartilages, whereas BMP overexpression with human
recombinant BMP4/7-soaked beads or Tg(hsp70I:Gal4;UAS:Bmp4)
causes dorsal cartilages to acquire more ventral rod-like shapes.
Based on their morphologies, we interpret these as dorsal-to-ventral
transformations of Ptp (an anterior subset of the dorsal domain)
(Talbot et al., 2010) and in some cases Pq (bead implantation in arch
1) or Hm (dorsal arch 2). These transformations correlate with
changes in gene expression – loss of BMP signaling reduces ventral
expression of hand2 and dlx3b, and expands jag1b expression
ventrally at 30 hpf (more than 24 hours prior to cartilage
differentiation), without disrupting NC migration or survival,
whereas BMP overexpression expands hand2 expression into the
dorsal domain in both arches 1 and 2. Thus, BMP signaling promotes
ventral identities in NC cells within the arches, and this may underlie
the ability of Bmp4 to promote incisor formation in mandibular
explants, and restrict Nkx3.2 expression to position the jaw joint
(Vainio et al., 1993; Thesleff and Sharpe, 1997; Tucker et al., 1998a).
Our results also suggest that BMPs act both directly on NC cells
and indirectly through induction of other signals in the mandibular
epithelium. Tg(Bre:GFP) is expressed in both the NC and the
surrounding epithelia at 16-24 hpf. Mosaic analyses suggest that
inhibiting Bmpr1a function in NC causes loss of hand2 and msxe
expression in a cell-autonomous manner (Zuniga et al., 2011).
However, BMP deficiency also causes a loss of edn1 and expansion
of fgf8a expression in the oral ectoderm, similar to previous studies
in mice (Stottmann et al., 2001), which would be predicted to alter
skeletal morphogenesis. Loss of Smad4 in NC cells also leads to
defects in Fgf8 expression in the adjacent oral ectoderm in mice (Ko
et al., 2007). Hence, our results are consistent with either direct
effects of BMP signaling on the ectoderm or feedback interactions
between NC and ectoderm.
DEVELOPMENT
Fig. 6. Exogenous Edn1 rescues BMP deficiency and vice versa. (A-D,F-K) Alcian Blue stained cartilages of 5 dpf larvae, anterior towards the
left. (A-D)Dissected cartilages, lateral views, of a control (A), Tg(hsp70I:dnBmpr1a-GFP) transgenic heat shocked at 16-18 hpf (B) and similarly
treated transgenics in which 0.5mg human recombinant EDN1 protein was microinjected into the arches at 25 hpf (C) and 31 hpf (D). (E)Histogram
depicting percentages of Tg(hs:dnBmpra1-GFP)+ embryos with severe ventral arch defects without and with EDN1 protein injected.
(F-H)Ventral view of neurocranial cartilage in control (F), dnBmpr+ (G) and dnBmpr+ with 0.5mg EDN1 injected (H). (I-K)Whole-mount cartilages,
ventral views, of non-transgenic wild-type control (I), non-transgenic edn1–/– mutant (J) and edn1–/–; Tg(hsp70I:Gal4;UAS:Bmp4) (K) embryos
subjected to a 1-minute heat shock at 21 hpf. Arrows show rescued ventral Mc; arrowheads show rescued ventral Ch cartilages; asterisks indicate
fused Mc-Pq joints. Ch, ceratohyal; e, ethmoid plate; Hm, hyomandibular; Mc, Meckels cartilage; Pq, palatoquadrate; Sy, symplectic; t, trabeculae.
Scale bars: 100mm.
BMP and Edn1 in craniofacial development
RESEARCH ARTICLE 5143
An integrated Edn1 and BMP signaling network in
DV patterning
BMP and Edn1 signaling initially play similar roles in promoting
ventral (distal) skeletal fates in the mandible (Aberg et al., 1997; Liu
et al., 2005; Clouthier et al., 2010). We argue that BMPs initially
promote Edn1, as heat-shocked dnBmpr transgenics lose edn1
expression, and restoring Edn1 partially rescues ventral fates and
cartilage patterning in the absence of BMP (Fig. 8A). However,
rescue occurs only during a brief early time window and clearly
some later roles for BMP signaling are Edn1 independent. This is
important, as there must be additional DV signals besides Edn1 to
explain why injection of EDN1 protein in an unlocalized manner
throughout the mandibular arch primordium rescues the lower jaw
in edn1–/– mutants (Miller et al., 2000). As bmp4 expression remains
restricted ventrally in the absence of Edn1, this could provide the
necessary ventralizing information to allow correct DV patterning.
This partial overlap in the ventralizing activities of BMP and
Edn1 signaling probably reflects not only temporal differences in
their inductive activities but also overlap in some transcriptional
targets and not others. Both promote expression of Msx1 (msxe in
zebrafish), which is expressed in the ventral arches [msxe actually
marks a distinct ventral-intermediate domain in zebrafish (Zuniga
et al., 2011) and is required for migration and survival of cranial
NC cells in mice (Ishii et al., 2005)]. However, the close relative
and direct BMP target Msx2 appears to be Edn1 independent
(Ruest et al., 2004). Both BMP and Edn1 induce expression of
ventrally expressed Dlx genes, including Dlx5 and Dlx6, which,
when deleted, cause ventral truncations and DV transformations of
the mandible that are strikingly similar to loss of Edn1 (Miller et
al., 2000; Ozeki et al., 2004). However, by 24 hpf, dlx3b in the
zebrafish becomes Edn1 dependent, but less sensitive to BMP.
One potential target of particular interest is Hand2, which has
been reported to respond quite differently to BMPs and Edn1, and
is crucial for establishing DV arch domains. Current models
suggest that Edn1 acts at short range (two or three cells) to induce
Dlx5/6, possibly through Mef2c (Miller et al., 2007), which in turn
induce Hand2 through its arch-specific enhancer (Charite et al.,
2001). Human recombinant BMP4-soaked beads do not induce
Hand2 expression in the chick embryo (Wilson and Tucker, 2004)
but Hand2 expression is reduced when Bmp4 is removed from arch
epithelia in mice (Liu et al., 2005). Our results in zebrafish suggest
that both BMPs and Edn1 are required for hand2 expression in
early arches (18-20 hpf), but BMPs are much more potent inducers
of hand2 just a few hours later. EDN1 overexpression also does not
rescue hand2 expression in the absence of BMP signaling despite
being able to restore some ventral cartilage and joint formation.
DEVELOPMENT
Fig. 7. Edn1 rescues ventral patterning in BMPdeficient embryos. (A-O)Whole-mount in situ
hybridization to detect expression of genes involved
in DV arch patterning at 30 hpf, lateral views,
anterior towards the left in controls (A,D,G,J,M),
Tg(hsp70I:dnBmpr1a-GFP) transgenics heat shocked
at 16-18 hpf (B,E,H,K,N) and heat-shocked
transgenics injected with human recombinant EDN1
protein (C,F,I,L,O). hand2 (A-C) expression is
unaffected, whereas dlx5a (G-I), dlx6a (J-L) and msxe
(M-O) expression are restored by EDN1 protein in
heat-shocked transgenics, and dlx3b (D-F)
expression is induced throughout the DV extent of
the arches. Arches 1 and 2 are numbered. Arrows
indicate rescued gene expression. Scale bar: 100mm.
5144 RESEARCH ARTICLE
Development 138 (23)
Another target gene that responds differently to BMPs and
Edn1 depending on the context is Nkx3.2. While Bmp4 has been
reported to inhibit Nkx3.2 in the mesenchyme in chick to restrict
it to the joint, we find that zebrafish nkx3.2 expression is
severely reduced in BMP-deficient zebrafish embryos. This is
more consistent with the hypothesis that BMPs initially promote
Nkx3.2 expression as well as other intermediate-specific Dlx
genes (Tucker et al., 1998a) and our results suggest that they
may do so indirectly through Edn1. Later, Hand2 represses
intermediate-specific genes such as dlx3b/4b/5a, thereby
restricting nkx3.2 to the joint domain, and opposes dorsal signals
such as Jag1b, thereby further refining domains along the DV
axis (Zuniga et al., 2010). Such intermediate-specific defects are
also consistent with previous studies of Ednra (Clouthier et al.,
2000; Nair et al., 2007) and would help explain the distinct
ventral and intermediate skeletal defects reported for Bmp2,
Bmp4 or Edn1 mutants.
This apparent contradiction may reflect the fact that we cannot
detect very low levels of rescued hand2, which are sufficient to
restore ventral skeletal identities. Alternatively, or in parallel,
BMPs may rescue other aspects of Edn1 deficiency, such as NC
proliferation and survival in the ventral arches. BMPs may also
help induce the subset of Hand2-expressing cells that remain in the
ventral-most arch in the absence of Edn1/EdnrA signaling, both in
fish and mice (Ruest et al., 2004; Nair et al., 2007). Such results
are also consistent with Hand2 being induced by BMPs in other
contexts, such as in precursors of sympathetic neurons and
endodermal cells of the gut (Howard et al., 2000; Wu and Howard,
2002).
Evolution of the D-V pharyngeal patterning
system
The collaboration between BMP and Edn1 that we describe here
for the pharyngeal skeleton may be an ancient and common motif
in vertebrates. Both signals are active and have been shown to
interact in smooth muscle and vascular endothelial cells (Dorai et
al., 2000; Bouallegue et al., 2007; Le Bras et al., 2010), NC cells
that contribute to the heart outflow tract (Clouthier et al., 1998;
DEVELOPMENT
Fig. 8. Model. Schematic of the primordia of arches 1 and 2 at 20 hpf
(A), 30 hpf (B) and cartilages at 6 dpf (C), lateral views, anterior
towards the left. (A)Pharyngeal epithelia (blue) express Bmp2/4/7 and
Edn1. Ventral/intermediate (pink/orange stripes) and dorsal (green)
domains are indicated within the arch mesenchyme. BMPs induce
Edn1, and both induce hand2 expression ventrally and repress jag1b
dorsally. (B)Ventral (pink), intermediate (light orange), dorsal (green)
and anterior (dark orange) domains are indicated at 30 hpf. BMPs
induce hand2 expression as well as Edn1 expression, which in turn
induces intermediate domain genes (arrows) but not hand2 at this
stage. BMP also promotes trabecular development from the anterior
maxillary domain (dark orange). (C)Defects in BMP or Edn1 signaling
lead to defects in the corresponding dorsal and ventral cartilages, joints
and trabecular cartilages that derive from these different domains.
A direct role for BMPs in palate development
One craniofacial structure that is more difficult to fit into this
model of nested DV patterning domains induced by BMPs and
Edn1 is the palate. Heat-shocked dnBmpr embryos lack the
trabecular cartilages of the primary palate, while BMP
overexpression leads to trabecular expansion/malformation. BMPdeficient mice often show palatal clefting. We and others have
previously shown that the trabeculae in zebrafish arise from NC
cells that lie within the maxillary region of the mandibular arch
(Wada et al., 2005; Eberhart et al., 2006). At 24 hpf, these cells are
located above the stomodeum and express Tg(Bre:GFP) (see Fig.
1). In addition, NC cells that form the Ptp process of the Pq
cartilage also lie in this region. Although inhibition of BMP
signaling in dnBmpr transgenics does not eliminate Ptp, BMP
overexpression appears to transform this process into a Mc-like
ventral element. Thus, BMPs may have conserved roles in
patterning the maxillary region of the first arch as well as the
palate. BMPs may act directly on these domains or through
expansion of fgf8a expression and other signals within the
stomodeum (an interesting issue to resolve in the future).
However, unlike DV patterning within the arches, we find no
evidence for overlapping functions of BMPs and Edn1 signaling in
trabecular development. Loss of Edn1 signaling has little effect on
the palate and restoring Edn1 does not rescue trabecular
development in BMP-deficient embryos. By contrast, in mice the
palatine bone, which also derives from the maxillary domain,
expands ventrally in mutants in the Edn1 signaling pathway (Miller
et al., 2000; Dudas et al., 2004; Ozeki et al., 2004; Liu et al., 2005).
In addition, when Edn1 is knocked into the EdnrA locus in mice,
it causes an apparent homeotic transformation of the maxilla into
a mandible (Sato et al., 2008) and misexpression of Edn1 induces
dlx3b/dlx5a in the maxillary domain in zebrafish (Zuniga et al.,
2010). Thus, our results suggest that while maxillary/palate
progenitors can respond to Edn1, they require BMPs in an Edn1independent manner in this more anterior craniofacial domain.
Yanagisawa et al., 1998a; Kaartinen et al., 2004), osteoblasts
(Guise et al., 2003; Canalis et al., 2005), and some forms of
prostate cancer (Dawson et al., 2006; Ye et al., 2007; Whyteside et
al., 2010). In the future it will be interesting to determine whether
or not a similar genetic hierarchy exists between BMP and Edn1,
and whether they regulate similar targets in these different contexts.
The DV patterning system of BMP and Edn1 signaling we
describe for the craniofacial skeleton also may have been pivotal
in jaw evolution. Recent evidence suggests that three DV domains
(ventral, intermediate and dorsal) arose before the divergence of
jawed vertebrates from their jawless ancestors, as living agnathans
(i.e. lampreys) show distinct DV-restricted domains of Dlx gene
expression, presumably corresponding to mandibular, intermediate
and maxillary domains (Neidert et al., 2001). This is despite the
absence of a jaw joint or focal expression of an Nkx3.2 ortholog in
the arches of these animals. Notably, however, Hand2 expression
is ventrally restricted in lamprey embryos (Kuraku et al., 2010).
Based on our data, this may have first arisen when Hand2
expression happened to fall under the control of an ancestral,
ventrally localized BMP or Edn1 signal. Subsequent reinforcement
of ventral-specific signaling through positive- and negativefeedback interactions between these two signals (as well as Notch
signaling) could have led to a robust DV patterning system. In
addition, divergence in target gene regulation by these two
ventralizing signals, together with cross-inhibitory interactions
between their targets may have further defined the intermediate
domain to give rise to joints. In this way, a primitive DV patterning
system in the chordate ancestors of vertebrates could be established
that was later refined to allow formation of a lower jaw. Further
refinement of this system may have been a crucial innovation in the
evolution of a biting jaw in gnathostomes.
Acknowledgements
We thank Ines Gehring, Megan Matsutani and Corey Gingerich for fish care,
Pam Lincoln and Patrick Hsieh for help with generating the Tg(Bre:GFP) line,
and David Kimelman for the Tg(hsp70I:dnBmpr1a-GFP) line.
Funding
This research was supported by the National Institutes of Health
[F31DE020248 to E.Z., R01HD056219 to K.W.C., R01DE018405 and
R01DE018405-S03 to J.G.C., R01DE13828 to T.F.S.]; and March of Dimes
[FY08-459 to J.G.C.]. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.067801/-/DC1
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DEVELOPMENT
5146 RESEARCH ARTICLE